rpc00009: Nicotiana tabacum T-13 cell suspension culture

Note

  1. Components
  • Domestic delivery: Two 50-mL tubes, containing 25 mL of cell suspension
  • Overseas delivery: Two 250-mL flasks, containing cells placed on semi-solid medium

2.Notice

  • Subculture the cells to fresh medium immediately after arrival.
  • Do not store the cell culture in a refrigerator and a freezer.
  • Maintain aseptic conditions of the cell culture, and work in a laminar flow cabinet.
  1. Summary
  • Culture medium: MS medium, pH 5.8 (medium no. 6)
  • Culture conditions: 27°C, dark, 120 rpm
  • Subculture: 7-day intervals
  1. Citation of cell line

    When results obtained by using this cell line are published in a scientific journal, it should be cited in the following manner: “Nicotiana tabacum T-13 cell line (rpc00009) was provided by the RIKEN BRC which is participating in the National BioResource Project of the MEXT/AMED, Japan.”

Introduction

Tobacco T-13 cell line was established from a stem of Nicotiana tabacum L. cultivar Bright Yellow (Hino et al. 1982 [1]). The T-13 cell culture has a high capacity for producing coumarin (scopolin). The T-13 cells are grown in a phytohormone-free Murashige and Skoog (MS) medium, pH 5.8. Our T-13 cell culture has been maintained in the dark at 27°C with rotary shaking at 120 rpm and subcultured at 7-day intervals.

Materials

Chemicals and stock solutions

(All stock solutions are stored at 4°C)
  • MS salt mix

    Murashige and Skoog Plant Salt Mixture, Wako Pure Chemical Industries (#392-00591)

  • Sucrose

  • MS_VT

    Chemical Concentration (mg/mL)
    Nicotinic acid 0.5
    Pyridoxine·HCl 0.5
    Thiamine·HCl 0.1
    Glycine 2
  • MS_inositol

    Chemical Concentration (mg/mL)
    myo-Inositol 40
  • KOH (1 N)

Glassware

  • Erlenmeyer flask (300 mL), capped with two layers of aluminum foil
  • Pipette (10 mL; large tip opening) and a bulb, sterilized by autoclaving at 121°C for 20 min

Preparation of MS medium (medium no. 6)

  1. Dissolve the following chemicals in approximately 800 mL of distilled water.

    Chemical Amount
    MS salt mix 1 bag (1 L)
    Sucrose 30 g
  2. Add following stock solutions, and fill up to approximately 950 mL with distilled water.

    Stock solution Volume (mL)
    MS_VT 1
    MS_inositol 2.5
  3. Adjust the pH of the solution to 5.8 with KOH (1 N), and fill up to 1 L with distilled water.

  4. Pour 75 mL of the medium into a 300-mL flask.

  5. Autoclave the flask at 121°C for 20 min.

Methods

  1. Leave to stand a flask containing a 7-day-old culture until the cells settle to the bottom of the flask.
  2. Transfer 10 mL of the sedimented cells to 75 mL of fresh MS medium with a pipette.
  3. Incubate cell cultures on a rotary shaker at 120 rpm under the dark condition at 27°C.

Notes

  • For domestic customers: We send T-13 cell suspension in 50-mL disposable tubes. The cells should be transferred to fresh MS medium immediately after arrival. Transfer settled cells to Erlenmeyer flasks containing fresh liquid medium with a pipette.
  • For overseas customers: We send T-13 cells placed on semi-solid MS medium in 250-mL disposable flasks. The cells should be transferred to fresh MS medium immediately after arrival. Collect the cells from the semi-solid medium with a spatula and transfer them to Erlenmeyer flasks containing fresh liquid medium.
  • In order to maintain T-13 cell suspension cultures stably, it is essential to transfer an adequate amount of cells to fresh MS medium in every subculture. The amount of cells may vary from one lab to another, because proliferation of T-13 cells is affected by culture conditions, such as a room temperature, rotation speed of a rotary shaker, and aeration condition of the culture.
  • In order to obtain good aeration of a suspension culture, a silicone cap may be used instead of the aluminum foil cap.

Maintenance history

References

[1]Hino F, Okazaki M, Miura Y (1982) Effect of 2,4-dichlorophenoxyacetic acid on glucosylation of scopoletin to scopolin in tobacco tissue culture. Plant Physiology 69: 810–813. DOI: 10.1104/pp.69.4.810